Method for detection of hepatocellar carcinoma (hcc) using an octanoate breath test

ABSTRACT

Methods and systems for diagnosis, prognosis and monitoring of hepatocellular carcinoma (HCC). Specifically, the present invention relates to the use of breath tests based on isotope-labeled octanoate in the detection and monitoring of HCC.

FIELD OF THE INVENTION

The present invention relates to diagnosis, prognosis, monitoring andtreatment of hepatocellular carcinoma (HCC). Specifically, the presentinvention relates to the use of breath tests based on isotope-labeledoctanoate in the detection and monitoring of HCC.

BACKGROUND OF THE INVENTION

Hepatocellular carcinoma (HCC, also referred to as malignant hepatoma)is a primary malignancy of the liver. HCC most commonly appears inpatients with chronic viral hepatitis (e.g. hepatitis B) and/orcirrhosis with any etiology. Non-alcoholic steatohepatitis (NASH) hasalso been found to be a risk factor for the development of HCC. Examplesof other risk factors include high aflatoxins exposure and iron overloadconditions such as hemochromatosis. In some cases, cryptogenic HCC isdeveloped in patients with no history of liver disease or known riskfactors.

HCC incidence worldwide is constantly increasing. In some parts of theworld, such as in sub-Saharan Africa, China, Hong-Kong and Taiwan, HCCis a major health problem, probably due to high exposure to hepatitisviruses like B and C and to regional exposure to environmentalpathogens.

The pathogenesis of HCC is not completely known, however mitochondrialdysfunction is suggested to be involved. HCC cells show genetic andmetabolic alterations with a decreased mitochondrial function (Warburg O(1956). “On the origin of cancer cells”. Science 123 (3191): 309-14).

Treatment of HCC may be directed towards a cure, or focused on relief ofsymptoms and prolongation of life. The type of treatment is typicallyselected according to the tumor size and stage. When the tumor is small(less than 2-3 cm), limited to one lobe of the liver, without evidenceof invasion of the liver vasculature and in a well preserved liverfunction, surgical resection may be performed. Other treatment optionsinclude liver transplantation, radiofrequency ablation (RFA) andtransarterial chemoembolization (TACE) (for treatment algorithms, see,for example, the Barcelona Clinic Liver Cancer (BCLC) Staging System).In RFA, radiofrequency energy is transmitted to the tumor via a needleelectrode which is advanced into the tumor under image guidance (such asX-ray screening, CT scan or ultrasound). The radiofrequency wavespassing through the needle lead to tumor destruction by thermalcoagulation and protein denaturation. In TACE, chemotherapy isadministered directly to the tumor via a catheter, and blood supply tothe tumor is cut-off. In addition to the procedures described above,oral medicines may also be administered. For example, sorafenib tosylate(Nexavar™), an oral medicine that blocks tumor growth by activating theintrinsic mitochondrial pathway (Kurosu, “Sorafenib induces apoptosisspecifically in cells expressing BCR/ABL by inhibiting its kinaseactivity to activate the intrinsic mitochondrial pathway”, CancerResearch 2009 May 1; 69(9):3927-36.), is approved for patients withadvanced HCC.

In general, small or slow growing tumors may be successfully treated ifdiagnosed early. However, early diagnosis is difficult, partiallybecause most of the patients who develop HCC have no symptoms other thanthose related to their longstanding liver disease. Surveillance ofhigh-risk groups, such as cirrhotic patients, is usually performed tofacilitate early detection of HCC (see, for example, AASLD Guidelines).

The detection and diagnosis of HCC is typically based on imaging tests,serology tests and sometimes biopsy. Imaging tests include, for example,abdominal ultrasound, helical computed tomography (CT) scan, triplephase CT scan and magnetic resonance imaging (MRI). Serology testsinclude measurement of blood levels of alpha-fetoprotein (AFP), wherehigh levels of AFP are associated with HCC. The typical strategy is6-monthly surveillance with AFP and ultrasound.

Assessment of HCC is also performed as part of post-treatmentmonitoring. Evaluation of treatment efficacy and determination of activevs. inactive HCC are usually performed radiologically usingcontrast-enhanced CT or MRI. Exemplary contrast media is Lipiodol®, aniodized oily agent that is selectively retained within the tumormicrovessels. Lack of vascular enhancement in the treated lesion istypically indicative of positive response to the treatment. AFP is notaccurate enough as a follow-up tool, and the monitoring of AFP levelsafter therapy does not replace imaging. The ideal imaging interval isunknown, but initially a 3-4 month interval is commonly used to monitorHCC lesions after initial treatment.

The standard techniques for HCC diagnosis and follow-up have severaldrawbacks. For example, imaging methods based on CT or MRI, areconsidered expensive, must be performed in a hospital by a skilledpractitioner, and associated with high radiation and side effects suchas contrast-media-induced nephropathy in the case of CT/MRI. Inaddition, methods such as AFP measurements are insufficiently sensitiveor specific.

Breath tests based on monitoring ¹³CO₂ levels, which is a by-product ofmetabolism of ¹³C-labeled substrates by the liver, have been proposed asa tool for evaluation of liver function. If the hepatic metabolism of atest compound results in the formation of carbon dioxide, and theappropriate carbon is labeled, the exhalation of labeled CO₂ (which ismeasurable, for example, in mass spectrometry or non-dispersive infraredanalyzer), reflects the hepatic clearance of the original labeledcompound and may be used to assess specific liver functions. Forexample, compounds metabolized by hepatocytes cytochrome P450 enzymesmay be used in the assessment of liver microsomal function. Exemplarycompound is methacetin. As another example, compounds that undergometabolism in liver mitochondria may be used in the assessment of livermitochondria function and may be used to detect certain liver conditions(see for example, Grattagliano et al. (2010) Eur J Clin Invest, 40 (9):843-850 and Portincasa et al. (2010) Clujul Medical, 83: 23-26).

One exemplary molecule is ketoisocaproate (KICA), a compound thatundergoes decarboxylation in liver mitochondria. Use of KICA breathtests in evaluation of liver function in HCC patients has been reported(Palmieri et al. (2009) Journal of Surgical Research 157, 199-207). Inthis study, the effect of two different HCC treatments on liver functionwas evaluated. Cirrhotic patients with and without HCC were testedusing, inter alia, KICA decarboxylation. At baseline, patients with HCChad significantly lower ¹³C-KICA breath test values compared withhealthy controls and cirrhotic patients without HCC. Minor butsignificant changes in ¹³C-KICA breath test values emerged betweencirrhotic patients without HCC and healthy controls (lower values wereobserved for the cirrhotic patients without HCC compared to healthycontrol). The patients were treated by either TACE or RFA, after which¹³C-KICA levels were again tested at day 1, day 30 and day 180. Theauthors summarized: “ketoisocaproate decarboxylation was unaffected byTACE but decreased after RFA (−27%, P<0.05)”, and concluded: “RFA notTACE appears to spare residual (microsomal) liver mass, but induces sucha transient stunning effect on mitochondrial function”.

Another exemplary molecule that is metabolized by liver mitochondria isOctanoate. Octanoate is a medium chain fatty acid that entersmitochondria and undergoes β-oxidation generating acetyl coenzyme A(acetyl-CoA). Acetyl-CoA enters the Krebs cycle and is oxidized to CO₂unless it is utilized for the synthesis of other energy-rich compounds.

WO 2007/054940, to the Applicant of the present invention, disclosesbreath test devices and methods for the evaluation of liver functionaland metabolic capacity or to assess liver health and/or degree of liverinjury. For example, a method of evaluating a liver condition isdisclosed, the method includes on-line monitoring a metabolic product ofoctanoic acid, a salt or a derivative of octanoic acid, in a subject'sbreath after administering to the subject isotope labeled octanoic acid,a salt or a derivative thereof. As another example, a device forevaluating a liver condition is disclosed, the device includes one ormore sensors adapted to monitor on-line an isotope level of a metabolicproduct of labeled octanoic acid, or a salt or a derivative thereof in asubject's breath and a controller adapted to sample measurements of theone or more sensors at a continuous mode. The method and device may beused in distinguishing between a non-alcoholic fatty liver andnon-alcoholic steatohepatitis conditions in a subject.

WO 2010/013235, to the Applicant of the present invention, disclosesbreath test devices and methods that may be used for the evaluation ofliver functional and metabolic capacity or to assess liver health and/ordegree of liver injury. For example, a method of detecting abnormalbeta-oxidation associated with insulin resistance or alcoholic liverdisease or non-alcoholic fatty liver disease or metabolic syndrome isdisclosed, the method includes monitoring a metabolic product ofoctanoic acid, a salt or a derivative of octanoic acid, in a subject'sbreath after administering to the subject isotope labeled octanoic acid,a salt or a derivative thereof.

The complexity of liver metabolism imposes challenges on assessing itsfunction, and therefore interpretation of breath test results is notalways straightforward. Examples of potential limitations for theinterpretation of breath test results include the presence ofconfounding variables (e.g. exercise), the typology of gastric emptyingkinetics, the hepatic first pass metabolism of the administeredsubstrates, and the presence of competing pathways of elimination andmetabolism of compounds. Additional factors that should be taken intoaccount include, for example, the presence of mitochondrial metabolismoccurring in organs other than the liver (extra hepatic metabolism, whenhepatic metabolism is of interest, would negatively impact testrelevance), possible dilution of exogenous labeled compound in a largerpool of unlabelled compound, and endogenous production of unlabelled CO₂which can vary substantially from subject to subject. Furthermore, thereare many factors that result in high intra- and inter-patientvariability, and different disease etiologies may impact differentfunctions of the liver and may result in a different breath testoutcomes.

There still remains a need for cost effective, accurate and simplemethods and systems for surveillance, detection, prognosis andmonitoring response to treatment of hepatocellular carcinoma.

SUMMARY OF THE INVENTION

The present invention provides methods and systems for improved HCCdetection, evaluation, and monitoring response to treatment, whichutilize octanoate or salts or derivatives thereof.

The present invention discloses for the first time that the level ofoctanoate metabolism by liver mitochondria, as reflected in breath testsusing isotope-labeled octanoate, correlates with the presence of activeHCC, and can be used to detect and monitor HCC. In particular, it is nowdisclosed that lower values of isotope-labeled octanoate breath test(OBT), for example ¹³C-octanoate breath test, are indicative of activeHCC. It was surprisingly found that significantly lower ¹³C-octanoatebreath test values are observed for subjects having active HCC incomparison to subjects having inactive HCC and control subjects with noHCC. Subjects having inactive HCC exemplified herein below haveundergone TACE treatment and were evaluated close after and up to 3months after the procedure. The observed results are surprising, interalia, in light of earlier publications stating that: “TACE did notinduce an early fall of mitochondrial function, as observed for RFA,suggesting no specific interference of this procedure with mitochondrialfunction.” (Palmieri et al. (2009) Journal of Surgical Research 157,199-207).

The present invention further discloses that OBT values may be used fordetection of HCC even in early stages. Remarkably, it was found thateven in a patient with HCC<2 cm, which represents a very small part ofthe liver mass, OBT measurements are significantly reduced. The presentinvention further discloses that OBT values may be used inpost-treatment follow-up of patients with HCC, for the assessment ofresponse to treatment and/or recurrence of the disease.

The methods and systems of the present invention may be applicable forscreening/surveillance and early detection of small and larger tumors.The methods and systems of the present invention may also be applicablefor monitoring the response to several types of HCC treatments, forexample, TACE and RFA treatments. OBT may also enable early detection ofrecurrence following resection of the tumor, or following livertransplantation.

According to one aspect, the present invention provides a method for HCCdetection, determining a prognosis and/or follow-up in a subject, themethod comprising:

(i) monitoring an isotope-labeled metabolic product of octanoate inexhaled breath of the subject following administration of anisotope-labeled octanoate;

(ii) comparing octanoate metabolism in the subject to a referenceoctanoate metabolism, wherein a significantly decreased octanoatemetabolism is indicative of active HCC.

In some embodiments, the method further comprises normalization of thebreath test values according to disease etiology, as detailed below. Inadditional or alternative embodiments, the method further comprisesnormalization of the breath test values according to a treatment, asdetailed below. In some embodiments, the method further comprisesdistinguishing between active and inactive HCC based on the comparison.

The method according to embodiments of the present invention is usefulfor early detection of active HCC, for example, HCC smaller than 2 cm.The decrease in OBT in patients with small active HCC (e.g. <2 cm),which could be masked by inter and intra variability of the metabolismby the great majority of the liver mass, cannot be profoundly explainedbased on the prior art. Without wishing to be bound by any particulartheory or mechanism of action, it may reflect a factor or factorssecreted by the tumor cells that is/are affecting the overallmitochondrial function in the liver of these patients. Any type oftherapy that counteracts this factor or these factors may have an effecton alleviation of the malignant process. Alternatively or additionally,it may be due to trapping of the octanoate in the tumor, for exampleinto its hypervascularity regions or in the tumor cells or in betweenthe cells and in vessels. The changes in vessels associated with thetumor might attribute to the enhancement of the effect beyond the directmass of the tumor. The potential trapping effect may serve as a mean fordelivering contrast agents or therapeutic agents to the tumor. Adelivery mechanism to HCC may enable to achieve high concentration of atherapeutic agent in the tumor without systemic side effects.

The present invention addresses some of the limitations of prior arttests, for example, ¹³C-KICA breath tests. As opposed to KICA used inprior art, octanoate metabolism is not affected by the overall liverfunction and remains normal in cirrhotic patients. Advantageously,octanoate breath tests allow a greater differentiation between patientswith impaired liver (due to fibrosis stage or cirrhosis) without HCC,that will have an octanoate metabolism similar to normal subjects andnot already decreased due to reduced liver reserve like in KICA, andthose with HCC.

As used herein, the term “octanoate” encompasses octanoate and salts orderivatives thereof, such as octanoic acid. The ¹³C-labeled “octanoate”is known by the generic name of sodium caprylate, sodium salt ofcaprylic acid. The IUPAC name for the sodium caprylate is1-¹³C-Octanoate Sodium. Additional synonyms include: 1-¹³C-OctanoateSodium; Sodium Octanoate, Octanoic Acid Sodium Salt, Sodium n-Octanoate,Sodium Octoate.

As used herein, “monitoring an isotope-labeled metabolic product ofoctanoate” refers to detecting and measuring a change in isotope ratioin exhaled breath of a subject over a predetermined period of time. Theterm “isotope ratio” refers to the ratio between the isotope selectedfor octanoate labeling and the naturally-abundant isotope. In someembodiments, monitoring is performed by continuous measurement over apredetermined period of time following a single administration oflabeled octanoate. In other embodiments, the isotope ratio is measuredin breath samples collected from the subject at periodic intervalsfollowing a single administration of labeled octanoate. According tothese embodiments, a plurality of samples is collected over apredetermined period of time.

In some typical embodiments, the metabolic product is CO₂. In someembodiments, the isotope is selected from the group consisting ofcarbon-13, carbon-14 and oxygen-18. In some typical embodiments, theisotope is ¹³C.

In some embodiments, comparing octanoate metabolism in the subject to areference octanoate metabolism comprises generating at least on of deltaover baseline (DOB) curve, percentage dose recovery (PDR) curve andcumulative PDR (CPDR) curve for the subject, and comparing at least oneparameter of said DOB, PDR or CPDR to at least one parameter ofreference DOB, PDR, CPDR or a combination thereof.

In some embodiments, comparing octanoate metabolism in the subject to areference octanoate metabolism comprises generating PDR curve andcomparing at least one parameter of said PDR curve to at least oneparameter of a reference PDR.

In some embodiments, the at least one parameter is selected from thegroup consisting of PDR maximum level (peak height), time of appearanceof the peak (time to peak) and the slope of rate of metabolism. Eachpossibility represents a separate embodiment of the invention.

In some specific embodiments, the parameter is peak height. According tothese embodiments, a decreased peak height is indicative of HCC.

In additional specific embodiments, the parameter is time to peak.According to these embodiments, a longer time to peak is indicative ofHCC.

In yet additional specific embodiments, the at least one parameter isone or more PDR values (% dose/hr) at selected time points. According tothese embodiments, a decreased PDR value at a selected time point isindicative of HCC.

In some specific embodiments, the at least one parameter is one or moreCPDR values at selected time points. According to these embodiments, adecreased CPDR value at a selected time point is indicative of HCC.

In some specific embodiments, the at least one parameter is one or moreDOB values at selected time points. According to these embodiments, adecreased DOB value at a selected time point is indicative of HCC.

In some typical embodiments, the labeled octanoate is administeredorally. In other embodiments it is administered intravenously or intranasally.

In some typical embodiments, the labeled octanoate is administered in apredetermined, single dose (e.g. 100 mg for each patient). In otherembodiments it is administered based on the patient's body weight (e.g.2 mg per kilogram).

In some embodiments, the method is adapted for follow-up and monitoringresponse to HCC treatment in a subject.

In some embodiments, the method comprises performing a first evaluationof the liver function by monitoring an isotope-labeled metabolic productof octanoate in exhaled breath of the subject following a firstadministration of an isotope-labeled octanoate, and performing a secondevaluation of the liver function after a predetermined period of time bymonitoring an isotope-labeled metabolic product of octanoate in exhaledbreath of the subject following a second administration of anisotope-labeled octanoate.

In some embodiments, the step of performing a second evaluation after apredetermined period of time is repeated a multiplicity of times.

It is to be understood that the terms “a first evaluation of the liverfunction” and “a second evaluation of the liver function” are notlimited to the initial evaluation session and the consecutive sessionthereafter, but may relate to any two separate evaluation sessions.

As used herein, “decreased”, “significantly decreased” or a “significantdifference”, typically refers to a statistically significant difference,as can be defined by standard methods known in the art.

In some embodiments, reference octanoate metabolism refers to a controloctanoate metabolism, as determined in control subjects not afflictedwith HCC. In some embodiments, the control subjects have at least onechronic liver disease without HCC. In some exemplary embodiments, thecontrol subjects are cirrhotic patients without HCC. In otherembodiments, the control subjects are healthy individuals with no liverdiseases.

In other embodiments, reference octanoate metabolism refers to octanoatemetabolism determined during a first evaluation of the liver function,e.g., octanoate metabolism previously measured in a tested subject. Forexample, if the method of the present invention is used for monitoring aresponse to treatment, breath test results obtained in a firstmeasurement from a particular subject may be used as reference forbreath test results obtained in a second measurement from the samesubject. For example, a first measurement may be performed before thebeginning of treatment and a second measurement may be performedfollowing a predetermined period of time after treatment. In this case,an increased octanoate breath test values in the second measurement incomparison to the first measurement are indicative of positive responseto treatment.

In some typical embodiments, the tested subject is a mammal, preferablya human.

In some embodiments, the tested subject is selected from the groupconsisting of a subject who is at risk of developing HCC, a subject whois suspected of having HCC, and a subject who is afflicted with HCC.Each possibility represents a separate embodiment of the invention.

In some embodiments, the subject is afflicted with HCC and has undergoneor is undergoing treatment. Each possibility represents a separateembodiment of the invention.

In some embodiments, the treatment is selected from the group consistingof TACE and RFA or any other drug for HCC including any type of tyrosinekinase inhibitors, and type of chemotherapeutic agent including but notlimited to Adriamycin, and any type of immunotherapy used for HCCincluding but not limited to pulsed dendritic cells, as well as anycombination of treatment. This also includes a follow up of response forany adjuvant treatment in patients undergoing surgical resection orliver transplantation. Each possibility represents a separate embodimentof the invention.

In some embodiments, the tested subject has at least one chronic liverdisease. In some embodiments, the chronic liver disease is selected fromthe group consisting of non-alcoholic steatohepatitis (NASH),non-alcoholic fatty liver disease (NAFLD), hepatitis B, hepatitis C orpatients with cirrhosis due to any etiology. In general, the disease maybe any other type of chronic liver disease with or without cirrhosis,including patients with idiopathic cirrhosis, exposing the patient toprimary liver cancer and/or to metastasis. Each possibility represents aseparate embodiment of the invention.

In some embodiments, the chronic liver disease is selected from thegroup consisting of NASH and NAFLD. Each possibility represents aseparate embodiment of the invention. According to these embodiments,the method further comprises normalization of the OBT values. NASH/NAFLDare known to affect the liver mitochondrial function (see, for example,Grattagliano I. et al., ¹³C-breath tests for clinical investigation ofliver mitochondrial function; Eur J Clin Invest 2010; 40 (9): 843-850).In some embodiments, an algorithm is used to correct the values ofNASH/NAFLD patients. In some embodiments, different cut-off values aredetermined for subjects with NASH/NAFLD.

In some embodiments, the tested subject has a liver disease other thanNASH. In some embodiments, the tested subject has a liver disease otherthan NAFLD. In some embodiments, subjects having a liver diseaseselected from the group consisting of NASH and NAFLD are excluded frombeing tested using the methods of the present invention.

In some embodiments, the tested subject is treated with sorafenib (whichis known to enhance mitochondrial function). According to theseembodiments, the method further comprises normalization of the OBTvalues. In some embodiments, an algorithm is used to correct the valuesof sorafenib-treated patients. In some embodiments, different cut-offvalues are determined for subjects treated with sorafenib.

This may also apply to any other type of therapy including anychemotherapy radiotherapy and adjuvant therapy, immunotherapy orinhibitors of intracellular mechanisms, or combination of the above.

In other embodiments, the subject is not treated with sorafenib or anyother cancer drug. In some embodiments, subjects treated with sorafenibor any other cancer drug are excluded from being tested using themethods of the present invention.

Thus, in some embodiments, the method further comprises normalization ofthe values obtained in step (i) according to disease etiology. Inadditional or alternative embodiments, the method further comprisesnormalization of the values obtained in step (i) according to atreatment.

In some embodiments, the method further comprises concomitant monitoringof total CO₂ in breath, for example, by capnography. This may enableminimizing test length and variations in metabolic rate and/or CO₂production that would introduce non-relevant variables to liver testevaluation.

These and further aspects and features of the present invention willbecome apparent from the figures, the detailed description, examples andclaims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Boxplot diagram and mean diamond representation of ¹³C-octanoatebreath test values in active vs. inactive HCC and control.

FIG. 2. ROC curve of ¹³C-octanoate breath test values in active vs.inactive HCC and control.

FIG. 3. ROC curve of ¹³C-octanoate breath test values in active vs.inactive HCC and control, excluding a sorafenib-treated patient.

FIG. 4. Boxplot diagram and mean diamond representation of ¹³C-octanoatebreath test values in active vs. inactive HCC and control, excludingNASH subjects with HCC and a sorafenib-treated patient.

FIG. 5. PDR curve of ¹³C-octanoate breath test of a 61 year old male HCVpatient without HCC.

FIG. 6. PDR curve of ¹³C-octanoate breath test of a 74 year old femaleHCV patient with active HCC.

FIG. 7. PDR curves of three consecutive ¹³C-octanoate breath tests of a69 year old female NASH patient with HCC, before and after treatmentwith relapse of HCC.

FIG. 8. PDR curves of three consecutive ¹³C-octanoate breath tests of a61 year old male with cryptogenic cirrhosis, before and after successfulHCC treatment.

FIG. 9. PDR curves of two consecutive ¹³C-octanoate breath tests of a 58year old male with HCV cirrhosis before and after successful HCCtreatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the use of isotope-labeledoctanoate or salts or derivatives thereof for the diagnosis, prognosisand follow-up of HCC.

The methods and systems of the present invention effectivelydifferentiate subjects with active HCC from subjects with inactive HCCor with no HCC. Thus, the methods and systems of the present inventionare useful for detecting, diagnosing and monitoring HCC in a subject.The methods and systems of the present invention may also be useful forHCC screening, detection of tumor progression, recurrence, prognosis andstaging. The methods of the present invention may also be useful forevaluating tumor size and response to any type of therapy. The methodsof the present invention may also be useful for detecting HCCrecurrence.

The methods of the present invention are based on analysis of breathtest parameters. Known quantities of an isotope-labeled exogenoussubstrate, namely isotope-labeled octanoate, are administered to asubject, and metabolism of the labeled substrate is quantitatively andqualitatively followed. The obtained breath test parameters are comparedto reference data. The obtained parameters are indicative of HCC statusin the subject. Breath tests according to embodiments of the presentinvention are based on detecting the isotope-labeled metabolic productin a subject's breath and measuring the ratio between labeled andunlabeled metabolic product. The data may be further processed, forexample, by calculating the rate of exhalation of the labeled metabolicproduct and generating a PDR curve.

Despite the difficulty in evaluating liver condition in breath tests,correlation was observed between the level of octanoate metabolism andthe status of HCC in a subject.

According to one aspect, the present invention provides a method forearly detection, prognosis and follow-up of HCC in a subject, the methodcomprising:

(i) determining octanoate metabolism in the subject by monitoring anisotope-labeled metabolic product of octanoate in exhaled breath sampleof the subject following administration of an isotope-labeled octanoate;and

(ii) comparing octanoate metabolism in the subject to a referenceoctanoate metabolism, wherein a significantly decreased octanoatemetabolism is indicative of HCC.

In some embodiments, there is provided herein a method for detection,determining the prognosis and/or follow-up of HCC in a subject, themethod comprising comparing octanoate metabolism in the subject asdetermined by monitoring an isotope-labeled metabolic product ofoctanoate in exhaled breath of the subject following administration ofan isotope-labeled octanoate to a reference octanoate metabolism,wherein a significantly decreased octanoate metabolism is indicative ofactive HCC.

In cirrhotic patients, overall liver function is impaired. Themetabolism of KICA, which is used in the prior art, is known to beaffected by the overall liver function, and impairment due to cirrhosisaffecting KICA breath test values and reducing them in comparison tonormal values in subjects with no cirrhosis. However, it is known thatsome of the HCC patients are not cirrhotic. It is therefore expectedthat a patient without cirrhosis, but with HCC cannot be efficientlydetected by KICA. Advantageously, the method according to embodiments ofthe present invention utilizes a compound whose metabolism is unaffectedby overall liver function. The OBT peak values are similar for healthy,controls with cirrhosis or subjects with inactive HCC versus thesubjects with HCC (independent of their actual liver impairment). It istherefore expected that a patient without cirrhosis, but with HCC can bedetected by octanoate breath test.

Typically, an isotope-labeled octanoate is administered to the subject,and breath sample(s) are collected. In some embodiments, monitoring isperformed by continuous measurement over a predetermined period of time.In some embodiments, a continuous measurement is performed using, forexample, a BreathID® System (Exalenz Bioscience Ltd.). Such measurementenables accurate assessment of the PDR peak, which is currently apreferred indicative measure for the presence of HCC, and may be usedfor early detection of small tumors.

In other embodiments, monitoring is performed by collecting a pluralityof breath samples from the subject at periodic intervals or at definedtime points over a predetermined period of time following a singleadministration of a labeled octanoate, and measuring the isotope ratioin said samples.

In some embodiments, samples are collected by continuous on-linesampling.

In some embodiments, the predetermined period of time ranges from about0.1-1 hour, from about 0.1-2 hours, from about 0.1-3 hours, from about1-2 hours, from about 1-3 hours, from about 1-4 hours, from about 2-4hours. Each possibility represents a separate embodiment of theinvention.

In some embodiments, a periodic interval for the collection of breathsamples ranges from about 0.5-30 min, from about 10-30 min, from about20-60 min, from about 30-60 min. Each possibility represents a separateembodiment of the invention.

In some embodiments, monitoring begins only after administration of thelabeled substrate. In other embodiments, monitoring begins before thelabeled substrate is administered. In some embodiments, a baselinereading or baseline values are generated.

In some embodiments, the exhaled isotope-labeled metabolic product ismeasured in at least three time points, for example, to generate apercentage dose recovery (PDR) curve. The metabolic activity may bedetermined from the PDR.

In some embodiments, the method includes on-line monitoring a metabolicproduct of octanoate in a subject's breath after administering to thesubject isotope-labeled octanoate.

In some typical embodiments, the metabolic product is CO₂. In someembodiments, the isotope is selected from the group consisting ofcarbon-13, carbon-14 and oxygen-18. In some typical embodiments, theisotope is ¹³C. For example, hepatic metabolism of ¹³C-octanoate may beassessed by measuring the ratio of ¹³C/¹²C in exhaled breath. Carbon-13is a stable, non-radioactive isotope, which can be incorporated into aspecific location within the molecule of a test substrate so that afterits metabolism by the liver and generation of ¹³CO₂, it would bereleased. The ¹³C-compound may be administered orally, rapidly absorbedand metabolized by the liver, and then the ¹³CO₂ may be measured inexhaled breath within a predetermined period of time.

In some embodiments, monitoring comprises generating at least one ofpercentage dose recovery (PDR) curve, cumulative percentage doserecovery (CPDR) curve and delta over baseline (DOB) curve, andcalculating at least one parameter of said PDR, CPDR and DOB curve.

In some embodiments, comparing comprises comparing the calculated atleast one parameter to at least one parameter of reference PDR, CPDR andDOB curves.

In some embodiments, comparing octanoate metabolism in the subject to areference octanoate metabolism comprises generating a percentage doserecovery (PDR) curve for the subject and comparing at least oneparameter of said PDR curve to at least one parameter of a reference PDRcurve. PDR curves are known in the art. Such curves depict the rate ofmetabolism of the labeled substrate in % dose/hour (percentage of theadministered dose recovered per hour), as measured in breath. PDR curvesreflect dynamic response of the liver.

In some embodiments, the at least one parameter is selected from thegroup consisting of peak height, time of appearance of the peak and theslope of rate of metabolism. Each possibility represents a separateembodiment of the invention. In alternative or additional embodiments,the parameter is one or more PDR values (% dose/hr) at selected timepoints.

In some embodiments, comparing octanoate metabolism in the subject to areference octanoate metabolism comprises generating a cumulativepercentage dose recovery (CPDR) curve for the subject and comparing atleast one parameter of said CPDR curve to at least one parameter of areference CPDR curve. CPDR curves are known in the art. Such curvesdepict the amount of the labeled substrate that was metabolized in %dose (cumulative percentage of the administered dose recovered overtime), as measured in breath. The cumulative recovery of labeled CO₂ inbreath can be calculated as the area under the curve (AUC) of PDR.

In some embodiments, the parameter is one or more CPDR values atselected time points, for example, CPDR values at 30, 40 and/or 45minutes.

In some embodiments, comparing octanoate metabolism in the subject to areference octanoate metabolism comprises generating a delta overbaseline (DOB) curve and comparing at least one parameter of said DOBcurve to at least one parameter of a reference DOB curve. DOB curves areknown in the art. Such curves depict the difference between the isotoperatio (for example, ¹³CO₂/¹²CO₂) in a test sample collected at a certaintime point and the corresponding ratio in a baseline sample.

In some embodiments, the parameter is one or more DOB values at selectedtime points.

PDR curves represent normalization of the DOB per subject taking intoconsideration the subject's CO₂ production rate based on height andweight and the amount of substrate administered. In some embodiments,the subject is administered a dosage of Octanoate based on the subject'sweight (e.g. 1 mg or 2 mg or 3 mg per kilo), According to theseembodiments, DOB curves are more preferred for analysis. In otherembodiments, the subject is administered a fixed, predetermined dose ofoctanoate (e.g., 100 mg). According to these embodiments, PDR curves aremore preferred for analysis.

Typically, the selection of breath test parameters for analysisaccording to embodiments of the present invention deals withextra-hepatic metabolism or overcomes the problem of extra-hepaticmetabolism. Generally, analysis is performed for information obtainedonly until a peak is detected, for example—peak height and peak time.

In some specific embodiments, the parameter is peak height. According tothese embodiments, a decreased peak height is indicative of HCC.

In some typical embodiments, the labeled octanoate is administeredorally, intravenously or intra-nasally.

In some embodiments, the method is adapted for follow-up and monitoringresponse to HCC treatment in a subject. In some embodiments, the methodcomprises performing a first evaluation of the liver function bymonitoring an isotope-labeled metabolic product of octanoate in exhaledbreath of the subject following administration of an isotope-labeledoctanoate, and performing a second evaluation, after a predeterminedperiod of time, of the liver function by monitoring an isotope-labeledmetabolic product of octanoate in exhaled breath of the subject. In someembodiments, the step of performing a second evaluation, after apredetermined period of time, is repeated a multiplicity of times.

The term “multiplicity” may refer to any number higher than 1. In someembodiment, the term “multiplicity” refers to any number higher than 2.In other embodiments, the term “multiplicity” refers to any numberhigher than 3.

As used herein, “decreased”, “significantly decreased” or a “significantdifference”, typically refers to a statistically significant difference,as can be defined by standard methods known in the art.

Typically, control octanoate metabolism is determined in subjects notafflicted with HCC. In some embodiments, the control subjects have atleast one chronic liver disease without HCC. In some exemplaryembodiments, the control subjects are cirrhotic patients without HCC. Inother embodiments, the control subjects are healthy individuals with noliver diseases.

Control octanoate metabolism, according to the principles of the presentinvention, is determined in at least one subject, preferably a pluralityof subjects. A set of control parameters determined in control subjectsmay be stored as a reference collection of data.

In some typical embodiments, the tested subject is a mammal, preferablya human.

In some embodiments, the tested subject is selected from the groupconsisting of a subject who is at risk of developing HCC, a subject whois suspected of having HCC, and a subject who is afflicted with HCC.Each possibility represents a separate embodiment of the invention.

In some embodiments, the subject is afflicted with HCC and has undergoneor is undergoing treatment. Each possibility represents a separateembodiment of the invention. In some embodiments, the treatment isselected from the group consisting of TACE and RFA or any other drug forHCC including any type of tyrosine kinase inhibitors, and type ofchemotherapeutic agent including but not limited to Adriamycin, and anytype of immunetherapy used for HCC including but not limited to pulseddendritic cells, as well as any combination of treatment. This alsoincludes a follow up of response for any adjuvant treatment in patientsundergoing surgical resection or liver transplantation. Each possibilityrepresents a separate embodiment of the invention.

In some embodiments, the method further comprises normalization of thevalues obtained in step (i) according to disease etiology. In someembodiments, the disease etiology is selected from the group consistingof NASH and NAFLD.

In alternative or additional embodiments, the method further comprisesnormalization of the values obtained in step (i) according to one ormore blood test results. In some embodiments, the one or more bloodtests are selected from the group consisting of fasting glucose levels,insulin levels, ALT levels, AST levels, ALP levels, GGTP levels,bilirubin levels, albumin levels and sodium levels.

In alternative or additional embodiments, the method further comprisesnormalization of the values obtained in step (i) according to an HCCtreatment that the subject is receiving or has received. In someembodiments, the treatment is sorafenib administration.

In some embodiments, the exhaled isotope-labeled metabolic product ismeasured spectroscopically, for example, by infrared spectroscopy, orwith a mass analyzer.

In some embodiments, monitoring the isotope-labeled metabolic product ofoctanoate in exhaled breath of a subject comprises the use of at leastone technique selected from the group consisting of gas-chromatography(GC), GC-lined mass-spectrometry (GC-MS), proton transfer reactionmass-spectrometry (PTR-MS), electronic nose device, and quartz crystalmicrobalance (QCM). Each possibility represents a separate embodiment ofthe invention.

In some embodiments, the tested subject has at least one chronic liverdisease. In some embodiments, the chronic liver disease is selected fromthe group consisting of non-alcoholic steatohepatitis (NASH),non-alcoholic fatty liver disease (NAFLD), hepatitis B and hepatitis Cor any other type of chronic liver disease with or without cirrhosisexposing the patient to primary liver cancer and/or to metastasis. Eachpossibility represents a separate embodiment of the invention.

In some embodiments, the chronic liver disease is selected from thegroup consisting of NASH and NAFLD. Each possibility represents aseparate embodiment of the invention. According to these embodiments,the method further comprises normalization of the OBT values. NASH/NAFLDare known to affect the liver mitochondrial function (see, for example,Grattagliano I. at al. ¹³C-breath tests for clinical investigation ofliver mitochondrial function; Eur J Clin Invest 2010; 40 (9): 843-850).

In some embodiments, an algorithm and/or a different cut-off is used tocorrect the values of NASH/NAFLD patients. A suitable algorithm mayinclude certain blood test results that represent the disease severityof those patients, including, but not limited to, fasting glucoselevels, insulin levels and/or liver panel (e.g. ALT, AST, ALP, GGTP,Bilirubin, Albumin, Sodium levels).

In some embodiments, the tested subject has a liver disease other thanNASH/NAFLD.

In some embodiments, the tested subject is treated with sorafenib(enhancing mitochondrial function). According to these embodiments, themethod further comprises normalization of the OBT values and/or use of adifferent cut-off. In some embodiments, an algorithm is used to correctthe values of sorafenib-treated patients. For example, the breath testresults may be normalized as a function of the sorafenib dosage,frequency, and time of therapy already completed.

This may also apply to any other type of therapy including anychemotherapy radiotherapy and adjuvant therapy, immunotherapy orinhibitors of intracellular mechanisms, or combination of the above. Inother embodiments, the subject is other than a subject treated withsorafenib,

The method may further include monitoring total CO₂ in breath. CO₂ maybe monitored, for example, by capnography. This may minimize test lengthand variations in metabolic rate and/or CO₂ production that wouldintroduce non-relevant variables to the HCC evaluation.

The method may further include analyzing at least one breath relatedparameter obtained by monitoring the metabolic product of octanoate incombination with at least one breath related parameter obtained bymonitoring total CO₂ in breath. The method may further include analyzingat least one breath related parameter obtained by monitoring themetabolic product of octanoate in combination with at least onephysiological and/or medical parameter. The physiological and/or medicalparameter may include age, gender, weight, height, blood relatedparameter, body mass index (BMI), waist circumference, medicationtherapy related parameter, background diseases or any combinationthereof. Each possibility represents a separate embodiment of theinvention.

In some embodiments, the octanoate breath test results are combined withdemographic and clinical data of the subject to generate a predictionscore.

In some embodiments, demographic parameters are also considered andevaluated.

In some embodiments, the method further comprises computing an hepaticimpairment score (HIS) based at least on a breath test related parameterand on a demographic parameter.

Non-limiting examples of demographic parameters include height, weight,age, gender, smoking habits, disease etiology, known information aboutcomplications, or any combination thereof. The demographic informationcan be used to:

(i) compensate for inter-patient factors that affect a breath test;and/or

(ii) deal with factors that affect disease and that, together withbreath test data, may allow provision of a reliable predication ofdisease severity and/or status. The information may relate to any one ormore items from the following list (and/or to any other relevantinformation): height and weight, age, gender, smoking habits, diseaseetiology, known information about complications (including but notlimited to, shunts, portal hypertension, encephalopathy, varices,variceal bleeding, abnormal blood test such as bilirubin, edema and/orascites, decompensated cirrhosis, consumption of certain drugs that mayimpact the metabolic path of octanoate) and common scores that assessliver disease severity such as the Child-Turcotte-Pugh (CTP), Model forEndstage Liver Disease (MELD) and/or Sodium MELD (Na-MELD) scores.

Information about computing an HIS score can be found in InternationalPatent Application Publication No. WO 2010/013235.

In general, detecting, monitoring, distinguishing, evaluating,measuring, differentiating, quantifying, and the like as referred toherein may be accomplished by any of the apparatuses, breath collectionsystems, analyzer units, calibration devices, algorithms and methodsdescribed herein, and/or, as non-limiting examples, by any of theapparatuses, breath collection systems, analyzer units, calibrationdevices, algorithms and methods disclosed in U.S. Pat. Nos. 6,186,958,6,491,643 and 6,656,127; and U.S Patent Application Publication Nos.2003/0216660 and 2001/0021815.

Additional non-limiting examples of devices suitable for the methods ofthe present invention are those described in International PatentApplication Publication Nos. WO 2007/054940 and WO 2010/013235.

Typically, a device suitable for the methods of the present inventioncomprises a breath test analyzer, including a very sensitive gasanalyzer, capable of measuring a ratio of two chemically identical gaseswith different molecular weights. The gas analyzer is capable ofmeasuring small quantities of isotopically labeled gas, which may bepresent in the breath of a subject.

In some embodiments, there are at least two modes of analyzing thebreath samples. The analyzer can either perform its analysis onindividual exhaled breaths, or it can perform its analysis on-line onmultiple samples of the patient's breath, continuously collected fromthe patient.

In some embodiments, the breath test analyzer includes a breath analysischamber, a breath inlet conduit for conveying exhaled gas from a patientto the breath analysis chamber; and a gas analyzer operative to analyzegas in the breath analysis chamber and to conduct the first analyzing ofgas exhaled by the patient.

In some embodiments, monitoring an isotope-labeled metabolic product ofoctanoate is performed by continuous measurement. In some embodiments,on-line monitoring is performed, in real time, whilst a subject iscontinuing to provide breath for subsequent analyses. Suitable devicesfor on-line monitoring may include, for example, one or more breathsensors adapted to monitor an isotope level within a metabolic productof labeled octanoate, or a salt or a derivative of octanoate, and acontroller adapted to on-line sample measurements of the one or moresensors at a continuous mode.

The device may be adapted to sample measurements of the one or moresensors at a continuous mode, while the subject is coupled to the deviceduring breath sampling, for example, through a nasal cannula. The devicemay be adapted to automatically collect and analyze breath samples.

The device may further include one or more breath sensors, such ascapnography sensors, adapted to monitor CO₂ in breath.

The device may further include a processor adapted to analyze at leastone breath related parameter obtained by monitoring isotope level withina metabolic product of a labeled substance, such as octanoate, incombination with at least one breath related parameter obtained bymonitoring CO₂ in breath. The processor may correct for changes in CO₂exhaled/production of a subject throughout the breath test.

In some embodiments, a portable office-based system may continuouslysense and collect exhaled breath and analyzes CO₂ in on-line inreal-time through a nasal cannula worn by the subject, and may enableevaluation of HCC status in real time, thereby providing a follow-upmethod in clinical hepatology. In some embodiments, such a test isdesigned to provide a sensitivity and accuracy required for accuratedetection of clinically relevant variations as small as 1/1000 in the¹³CO₂/¹²CO₂ ratio.

In some embodiments, breath tests according to embodiments of thepresent invention are performed at the point-of-care.

Without wishing to be bound by any theory or mechanism of action, insome embodiments, the decrease in OBT in patients with active HCC, evenfor the ones smaller than 2 cm, may reflect either a factor or factorssecreted by the tumor cells that is affecting the overall mitochondrialfunction in these patients.

In some embodiments, any type of therapy that counteracts this putativefactor or factors, may have an effect on alleviation of the malignantprocess.

Without wishing to be bound by any theory or mechanism of action, insome embodiments, there is trapping of the octanoate in the tumor eitherdue to its hypervascularity or within the tumor cells or in between thecells.

In some embodiments, both mechanisms, trapping of octanoate and factoror factors secreted by the tumor cells may be acting simultaneously.

In some embodiments, the octanoate can be conjugated to achemotherapeutic agent as a method of delivering the chemotherapy intothe tumor, thus preventing or reducing unwanted systemic side effects ofthe drug, and enabling the use of high concentration of the drug insidethe tumor.

In some embodiments, these therapeutic agents also include any type ofchemotherapy, radiotherapy including but not limited to radioactivesubstrates such as iridium, immunotherapies, gene therapy, or anycombination of the above. The octanoate can therefore serve as an agentor carrier for delivering the therapy.

In some embodiments, since the octanoate is concentrated in the tumor itcan also serve as a tool for imaging, as an effective alternative tocurrently known agents, e.g. in cases where lipiodol is used in CT orMRI.

The methods of the present invention are based on metabolism ofoctanoate (C₈H₁₆O₂) by liver mitochondria. The metabolism of fatty acids(such as octanoate) and the release of the ¹³C-carbon in a form of ¹³CO₂requires multiple steps including beta-oxidation, generation of ¹³Clabeled Acetyl-CoenzymeA (AcCoA) and subsequently release of the ¹³Ccarbon in the tricarboxylic acid (TCA) cycle (also known as the citricacid cycle or the Krebs cycle). Improper TCA function may lead toaccumulation of AcCoA. It is known that alternative pathways exist forAcCoA, which result in ketone bodies generation or lipogenesis, whichwould not be detected in a breath test. The percentage of the labeledoctanoate that continues in the TCA cycle versus the percentage of thelabeled octanoate that goes to generation of ketone bodies may depend onthe physiological condition of the subject. For example, instarving/fasting conditions, oxalacetic acid may be needed (as it isused by the cells in the glucose synthesis/gluconeogenesis) whichresults in a less effective TCA process. The varying (and sometimesunpredicted) ratio between the amount of labeled octanoate that “takes”the TCA cycle path and the amount of labeled octanoate that “takes”alternative paths may affect the accuracy of the breath test. In someembodiments, the following steps are provided, independently from eachother or in any combination, for increasing the diagnostic accuracy ofthe octanoate breath test:

a. Using low dosage (such as in the range of about 100 mg) of octanoateor octanoate salt to avoid saturation of the TCA cycle. In general,octanoate dosage to be administered may be selected by body weight,e.g., about 1 mg/kg-3 mg/kg, allowing a dosage ranging from about 15 mg(in children with 1 mg/kg) to about 450 mg (in obese patients with 3mg/kg).

b. Patients may be tested after >8 hours fasting that assure that themetabolic conditions are more or less stable and less sensitive tovariations which are due to consuming a meal.

c. The test meal may include glucose and 13C octanoate.

d. The test meal may include aspartame (and 13C octanoate salt), whichprovides aspartic acid, which is the source of oxalacetic acid.

e. An alternative to c and/or d is wherein glucose/aspartame areadministered prior to the test.

f. Using of drugs that block/reduce the ketonic generation path-way (forexample, HMG-CoA reductase inhibitors).

g. Measuring ketone bodies generation with biochemical tests (ketonuriaand/or plasma serum ketone bodies concentration) in conjunction to the13C-octanoate breath test to improve diagnostic accuracy of the test.

h. Looking for traces of 13C-octanoate in blood.

According to another aspect, the present invention provides a device fordetection, prognosis and/or follow-up of HCC in a subject, the devicecomprising a processor configured to detect differences betweenoctanoate metabolism in the subject and control octanoate metabolism,wherein a decreased octanoate metabolism is indicative of HCC.

In some embodiments, the processor is configured to monitor anisotope-labeled metabolic product of octanoate in exhaled breath of thesubject following administration of an isotope-labeled octanoate, andcompare octanoate metabolism in the subject to a reference octanoatemetabolism, wherein a significantly decreased octanoate metabolism isindicative of active HCC.

In some embodiments, the processor is adapted to normalize the breathtest values according to disease etiology, treatment or a combinationthereof.

In some embodiments, the processor is adapted to calculate and generateat least one of DOB curve, PDR curve and CPDR curve, and compare atleast one parameter of said DOB, PDR or CPDR at least one referenceparameter.

In some embodiments, the at least one parameter is selected from thegroup consisting of PDR maximum level (peak height), time of appearanceof the peak (time to peak) and the slope of rate of metabolism. Eachpossibility represents a separate embodiment of the invention.

In some embodiments, the processor is further adapted to compute anoutput indication related to HCC based on the differences in octanoatemetabolism.

In some embodiments, the processor is further adapted to compute anoutput indication related to HCC based on the differences in at leastone parameter of DOB, PDR and/or CPDR.

In some embodiments, the processor is further adapted to concomitantlymonitor total CO₂ in breath.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES Example 1: 13C-Octanoate Breath Test Values in Active Vs.Inactive HCC and Control

Patients with active or inactive HCC (after successful therapy) wererecruited. The degree of activity was determined based on injection ofcontrast media (Lipiodol®) under CT and/or MRI. In addition, cirrhoticpatients without any evidence of HCC were also recruited (used as acontrol group). Information about the study population is provided inTables 1, 2 and 3 hereinbelow.

TABLE 1 Gender Active HCC Inactive HCC Control Grand Total F 4 3 4 11 M11 3 4 18 Grand Total 15 6 8 29

TABLE 2 Etiology Active HCC Inactive HCC Control Grand Total Cryptogenic 2* 1 — 3 HBV 2 1 1 4 HCV 7 3 6 16 NASH 4 1 — 5 Sarcoidosis — 1 1 GrandTotal 15  6 8 29 *One male patient was on Nexavar ™ (sorafenib)treatment before doing OBT.

TABLE 3 AFP levels HCC Status Minimum Maximum Median Mean Control 1.4233.30 3.40 7.39 Inactive HCC 4.60 31.90 6.28 12.27 Active HCC 3.995990.00 57.70 652.08

All patients have undergone dynamic ¹³C-octanoate breath test (OBT)using BreathID® device (Exalenz Bioscience Ltd.) before and/or aftertreatment. Three male subjects with active HCC, of which two with HCVand one with NASH, were tested twice (one test before treatment and onetest after).

The breath tests were performed according to the following procedure:

a. Preparation of the Study Subject:

-   -   Patients were asked to perform the breath test after an        overnight fast (including morning medication). The patients were        allowed to drink small amounts of water until 1 hour prior to        test. The patients rested for 3-5 minutes prior to the test        start (to assure that breathing rate and pulse are normal and        constant throughout the test).

b. Preparation of ¹³C-Octanaote:

-   -   100 mg of ¹³C-Octanoate powder were emptied into a disposable        cup and 150 cc of water were added. The mixture was mixed until        the substrate has been completely dissolved.    -   Just prior to the examination, this solution was poured into a        disposable cup.

c. Administration of the Breath Test:

-   -   i. Each patient was asked to sit in a chair in the room where        the test was performed.    -   ii. A nasal cannula was attached to a BreathID® device and to        the patient.    -   iii. The BreathID® device was activated and collected the        patient's baseline exhaled CO₂ for approximately 2 minutes.    -   iv. The patient was then instructed by the medical staff and by        an indication on the device to drink the test substrate.    -   v. The patient remained seated in the chair, breathing in a        normal manner for the next 60 minutes.    -   vi. The BreathID® device continuously measures and analyzes the        patient's exhaled breath in real time. As the test substrate is        metabolized, the value of the ¹³CO₂/¹²CO₂ ratio changes and        calculated in real time by the BreathID® system from the exhaled        breath. The BreathID® also calculates in real time the        percentage dose recovery (PDR), expressed in %/hour and the        cumulative PDR (CPDR). These values are displayed on the screen        of the BreathID® device as they are calculated in real time.    -   vii. If at any time the device does not detect patient's breath,        or if there is any other deviation from the desired test        requirements, the device produces an appropriate warning signal.    -   viii. At the completion of the procedure the nasal cannula was        removed and the patient was allowed to leave the testing room.

The patient was under the supervision of the physician or any otherqualified medical staff during the entire test.

For each breath test, a percentage dose recovery (PDR) and cumulativePDR (CPDR) curves were generated. The PDR peak values were groupedaccording to active/inactive HCC and control, and presented in a boxplotdiagram. Mean diamond representation was also generated. The results areshown in FIG. 1.

The data was entered into a validated excel sheet and analyzed with theAnalyze-It® Software version 2.12.

In addition, a receiver operating characteristic (ROC) curve wasgenerated (FIG. 2) and AUC_(ROC) was calculated in order to assess thepredictive value of the PDR peak in discriminating between active andinactive HCC or control. An AUC_(ROC) value of 0.89 (95% CI 0.74-1.00,p<0.0001) was obtained.

The outlier of the boxplot diagram (in FIG. 1) corresponds to a sampleobtained from a patient that was treated with sorafenib due to thepresence of HCC and prior to the OBT tests. Previous studies (see, forexample, Kuroso et al. (2009) Cancer Res. 69:3927-3936) have shown thatsorafenib can improve mitochondrial function. Accordingly, OBT valuesare expected to be higher in HCC patients treated with sorafenib. Asecond analysis of the results was performed, this time without thesample obtained from the sorafenib-treated patient. The ROC curve isshown in FIG. 3 showing an AUC_(ROC) value of 0.95 (95% CI 0.86-1.00,p<0.0001).

It is known from previous studies that the presence of NASH enhances OBTvalues (see for example, Braun M et al. “The unique breath ID testsystem diagnoses and predicts the extent of hepatic injury in patientswith nonalcoholic fatty liver disease”, Hepatology, 2005; 42: 752A.).Indeed, in the present study the samples of two NASH subjects with HCChad elevated OBT results. A third analysis was performed, this timewithout the samples obtained from the NASH subjects with HCC and withoutthe patient treated with sorafenib. The boxplot was regenerated fromthis cleaner sample (see FIG. 4). The ROC analysis has shown anAUC_(ROC) value of 1 (p<0.0001), meaning that full discriminationbetween active and inactive HCC or control can be obtained whenanalyzing the dataset without NASH and sorafenib treated subjects. Thisobservation suggests that for diagnostic purposes the etiology should beincluded (e.g. with or w/o NASH). Suggested cutoff w/o NASH is Peak26%/h (see dashed line in FIG. 4).

As noted above, three male subjects (two with HCV and one with NASH)were tested twice (once before treatment and once after). Breath testvalues of these subjects showed full agreement between OBT and patientstatus:

Subject 01—with Successful Treatment

First visit showed OBT peak=16.30%/h

Second visit 3 months after TACE showed OBT peak=31.78%/h

HCC became inactive following TACE (AFP was 5.84 and 5.48 ng/mLrespectively, demonstrating the sensitivity limitations of AFPmeasurements. Typically, AFP values above 10 ng/mL are consideredabnormal. Changes within the normal limits (9 and below) cannot bedetermined and evaluated accurately).

Subject 02—HCC Remained Active after TACE

First visit showed OBT peak=25.36%/h

Second visit 3 months after TACE OBT peak=22.19%/h

Clinically patient deteriorated and AFP increased from 3500 to 4800ng/mL.

Subject 05—NASH Patient with Successful Treatment after TACE

First visit showed OBT peak=30.35%/h

Second visit 5 months after TACE OBT peak=37.63%/h

Clinically patient improved and CT showed inactive HCC.

It was observed that the activity of HCC can be also determined for thetreatment follow-up. The NASH subjects may have another threshold todetermine the activity status of HCC, however the OBT Peak improved inNASH patient after successful treatment.

A summary of OBT performance parameters obtained from different analysesis provided in Table 4 hereinbelow.

TABLE 4 Populations N AUC CI P-value All 29 (15/14) 0.89 0.74-1.00<0.001 (active/inactive + control) Active/inactive 21 (15/6) 0.900.76-1.00 <0.001 Active/control 23 (15/8) 0.88 0.72-1.00 <0.001 All w/oNexavar ™ 28 (14/14) 0.95 0.86-1.00 <0.001 All w/o Nexavar ™ and 23(10/13) 1.00 NA NA NASH

As mentioned above an AUC_(ROC) value of 1 (p<0.0001), indicating fulldiscrimination between active HCC and inactive HCC+ control, wasobtained when the dataset was analyzed without Nexavar™ and withoutNASH.

Example 2: PDR Curves

Additional ¹³C-octanoate breath tests (OBT) were carried out and theirresults are shown in FIGS. 5-9. The tests were performed according tothe procedure described in Example 1 above.

FIG. 5 shows an OBT PDR curve of a typical cirrhotic patient withoutliver cancer. The PDR peak is reached within 30 minutes and isrelatively high (˜30%/h).

FIG. 6 shows an OBT PDR curve of a typical cirrhotic patient with livercancer. The PDR peak is delayed (after 30 minutes) and is typically low.

FIG. 7 shows PDR curves of three consecutive OBTs of a typical cirrhoticpatient with liver cancer. All curves are low. Although TACE treatmentshowed some improvement in curve no. 2 (“2^(nd) OBT treatment afterpartially successful treatment”) the relapse of HCC can be clearly seenon the 3^(rd) curve (“3^(rd) OBT 1 month after previous test showsrelapse of HCC”).

FIG. 8 shows PDR curves of three consecutive OBTs of a typical cirrhoticpatient with liver cancer who had a successful TACE treatment. Twocurves before treatment are low. The third curve shows characteristicsof a non-HCC case (high PDR Peak) and therefore points to the fact thatthe HCC treatment was successful. The lesion remained at 7 cm withoutgrowth and was in-active.

FIG. 9 shows PDR curves of two consecutive OBTs of a typical cirrhoticpatient with liver cancer who had a successful TACE treatment. The curvebefore treatment is low. The second curve shows characteristics of anon-HCC case (high PDR Peak) and therefore points to the fact that theHCC treatment was successful.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without undue experimentation and withoutdeparting from the generic concept, and, therefore, such adaptations andmodifications should and are intended to be comprehended within themeaning and range of equivalents of the disclosed embodiments. It is tobe understood that the phraseology or terminology employed herein is forthe purpose of description and not of limitation. The means, materials,and steps for carrying out various disclosed functions may take avariety of alternative forms without departing from the invention.

What is claimed is:
 1. A device for hepatocellular carcinoma (HCC)detection comprising a processor configured to: obtain a level of anisotope-labeled octanoate metabolic product measured in a subject'sbreath; compare the measured level of the isotope-labeled octanoatemetabolic product to a reference level; and detect HCC based on adifference between the measured level of the isotope-labeled octanoatemetabolic product and the reference level; wherein a significantlydecreased level of isotope-labeled octanoate metabolic product isindicative of active HCC.
 2. The device of claim 1, wherein theprocessor is further configured to distinguish between impaired liverfunction caused by fibrosis or cirrhosis and HCC, based on thecomparison.
 3. The device of claim 1, wherein the reference level is alevel of isotope-labeled octanoate metabolic product typically measuredin breath of healthy subjects.
 4. The device of claim 1, wherein thedevice further comprises a sensor configured to measure the level of theisotope-labeled octanoate metabolic product in the subject's breath. 5.The device of claim 4, wherein the sensor to configured to performcontinuous measurements.
 6. The device of claim 1, wherein theisotope-labeled octanoate is selected from the group consisting ofcarbon-13, carbon-14 and oxygen-18.
 7. The device of claim 1, whereinthe processor is configured to generate at least one of a delta overbaseline (DOB) curve, a percentage dose recovery (PDR) curve and/or acumulative PDR (CPDR) curve, and to compare at least one parameter ofsaid DOB, PDR or CPDR curves to at least one parameter of reference DOB,PDR and/or CPDR curves.
 8. The device of claim 7, wherein the processoris configured to generate a PDR curve and to compare at least oneparameter of said PDR curve to at least one parameter of a reference PDRcurve, wherein the at least one parameter is selected from the groupconsisting of PDR maximum level (peak height), time of appearance of thepeak (time to peak) and the slope of rate of metabolism.
 9. The deviceof claim 8, wherein the parameter is peak height and/or time to peak,and wherein decreased peak height is indicative of HCC and a longer timeto peak is indicative of HCC.
 10. The device of claim 1, wherein theprocessor is configured to obtain a disease etiology of the subject andto normalize the measured level of isotope-labeled octanoate metabolicproduct according to the disease etiology.
 11. The device of claim 10,wherein the disease etiology is NASH or NAFLD.
 12. The device of claim1, wherein the processor is further configured to obtain one or moreblood test results and to normalize the measured level ofisotope-labeled octanoate metabolic product according to the one or moreblood test results.
 13. The device of claim 12, wherein the blood testresults are selected from the group consisting of: fasting glucoselevels, insulin levels, ALT levels, AST levels, ALP levels, GGTP levels,bilirubin levels, albumin levels and sodium levels.
 14. The device ofclaim 1, wherein the processor is further configured to obtain atreatment that the subject is receiving or has received and to normalizethe measured level of isotope-labeled octanoate metabolic productaccording to the treatment.
 15. The device of claim 14, wherein thetreatment is sorafenib.
 16. The device of claim 1, wherein the processoris further configured to obtain values indicative of CO₂ levels in thesubject breath.
 17. The device of claim 1, further comprising a CO₂sensor.